Iodinated neuroprobe for mapping monoamine reuptake sites

ABSTRACT

An iodinated neuroprobe is provided for mapping monoamine reuptake sites. The iodinated neuroprobe is of the formula: ##STR1## wherein: R=aryl, substituted aryl, heterocyclic, CO(CH 2 ) n  Y, (CH 2 ) n  CHF 2 , and (CF 2 ) n  Y, wherein: 
     Y=Cl, Br, I, (CH 2 ) m , aryl, substituted aryl, heterocyclic CO 2  H, CO 2  R 3 , CO 2  NR 3  R 4 , OH, OR 3 , CH(OR 3 ) 2 , CR 3  (OR 4 ) 2 , OC0R 3 , OSO 2  R 3 , OCONR 3  R 4 , OCOOR 3 , CONR 3  R 4 , NR 3  R 4 , NR 3  COR 4 , NR 3  CO 2  R 4 , NR 3  CONR 4  R 5 , NCS, NCO; 
     R 3 , R 4  and R 5  =alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, or heterocyclic; 
     m=3-8; and 
     n=1-6; 
     R&#39;=C w  H 2w+1  wherein w=0-6 and C includes an isotope of carbon; and 
     X=an isotope of Cl, an isotope of Br, an isotope of F, an isotope of I, or Sn(R&#34; 1  R&#34; 2  R&#34; 3 ), wherein 
     R&#34; 1  =a C p  H 2p+1  group where p=1-6, or an aryl group; 
     R&#34; 2  =a CH p+1  group where p=1-6, or an aryl group; and 
     R&#34; 3  =a C p  H 2p+1  group where p=1-6, or an aryl group. 
     Related analogs are also provided. Additionally, a precursor of a radiolabeled neuroprobe and a kit for preparing the iodinated neuroprobe are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This Application is a continuation-in-part application of applicationSer. No. 08/185,689, filed Jan. 24, 1994, now U.S. Pat. No. 5,439,666,which is a continuation of application Ser. No. 07/841,617, filed Feb.25, 1992, now U.S. Pat. No. 5,310,912.

FIELD OF THE INVENTION

This invention relates to neuroprobes for mapping monoamine reuptakesites in the brain, and particularly to a neuroprobe that can also serveas a radiotracer for use in single-photon emission computed tomography(SPECT) and positron emission tomography (PET) for imaging of suchreuptake sites.

BACKGROUND OF THE INVENTION

A brain consists of a plurality of neurons that interact by exchangingchemical messengers. Each neuron generates neurochemicals, referred toas neurotransmitters; neurotransmitters act at sites on the cellularmembrane of a neuron, the sites being referred to as receptors.Receptors are associated with either ion channels through the cellularmembrane or secondary neurochemical messenger systems. By contrast,reuptake sites are molecular complexes which transport chemicals acrossthe cellular membrane of a neuron. When a neurotransmitter has servedits function, it is removed from the vicinity of the receptor by beingbound to a reuptake site which transports the neurotransmitter to theinterior of the neuron.

Just as there are many specialized neurons in the brain, there are alsoa variety of neurotransmitters, associated receptors, and reuptakesites. The distribution of specialized neurons depends upon theparticular organism under study, and the state of health of thatorganism.

A neuron can be classified according to the type of neurotransmitterthat it uses to communicate with other neurons. Certain types of neuronscan be found predominantly in particular regions of the brain. Forexample, the striatal region of a mammalian brain is innervated byneurons using dopamine as a neurotransmitter. The striatum also containsa large number of non-dopaminergic neurons that have dopamine receptors.Certain compounds, such as cocaine, have a preferential affinity fordopamine reuptake sites, and therefore tend to bind to such reuptakesites. The effect of a molecule such as cocaine upon a dopamine reuptakesite is to inhibit reuptake of the neurotransmitter dopamine, leavingmore dopamine available in the vicinity of the dopamine receptors.

In certain neurological diseases, such as Parkinson's disease, distinctgroups of neurons lose their normal physiological functioning.Consequently, the abnormal neurons may behave differently in thepresence of some neurotransmitters, and may also produceneurotransmitters in a manner that differs from a healthy neuron.

The major neurotransmitters, dopamine, norepinephrine, and serotonin,are referred to collectively as the monoamine neurotransmitters. Manyneurons have receptors adapted to receive at least one of theseneurotransmitters. Parkinson's disease is caused by the degeneration ofsome of the dopaminergic neurons in the brain. The neurons lost inParkinson's disease have a large number of dopamine reuptake sites;cocaine and chemical analogs of cocaine have an affinity for suchreuptake sites.

A radioisotope is commonly incorporated in molecules that have ademonstrated binding affinity for a particular type of neuroreceptor,and such molecules are commonly used as neuroprobes. The localization ofneuroprobes can be used to find specialized neurons within particularregions of the brain. It is also known that a neurological disease canbe detected by observing abnormal binding distributions of a neuroprobe.Such abnormal binding distributions can be observed by incorporating aradionuclide within each molecule of the neuroprobe with a high bindingaffinity for the particular reuptake sites of interest. Then, an imagingtechnique can be used to obtain a representation of the in vivo spatialdistribution of the reuptake sites of interest.

In single photon emission computed tomography (SPECT) imaging, the mostcommonly used radionuclides are heavy metals, such as ^(99m) Tc. Heavymetals are very difficult to incorporate into the molecular structure ofneuroprobes because such probes are relatively small molecules(molecular weight less than 400).

In positron emission tomography (PET), the radiohalide ¹⁸ F (fluorine)is commonly used as a substitute for H (hydrogen) inradiopharmaceuticals because it is similar in size. Not all halogenswill work, however. For example, I (iodine) is much larger than both Hand F, being approximately half the size of a benzene ring. However, dueto the small size of typical radiopharmaceuticals for use asneuroprobes, the presence of iodine markedly changes the size of thecompound, thereby altering or destroying its biological activity.

In addition, the presence of iodine in a neuroprobe tends to increaseits lipophilicity, and therefore increases the tendency of theneuroprobe to engage in non-specific binding. For example, paroxetine isa drug with high affinity and selectivity for serotonin reuptake sites,and ³ H!paroxetine has been shown in rodents to be a useful in vivolabel (Scheffel, U. and Hartig, P. R. J. Neurochem., 52: 1605-1612,1989). However, several iodinated analogs of this compound with iodineattached at several different positions had unacceptably low affinity,in fact being one tenth of the affinity of the parent compound.Furthermore, when the iodinated compound was used as an in vivoradiolabeled neuroprobe, non-specific binding activity was found to beso high that no measurable portion of the brain uptake appeared to bespecifically bound to the serotonin reuptake site. Thus, the iodinatedform of paroxetine is not useful as an in vivo probe.

The addition of iodine to a neuroprobe can unfavorably alter itsbiological properties. For example, tomoxetine has high affinity andselectivity for norepinephrine reuptake sites. However, when tomoxetineis iodinated, e.g. to form R-4-iodotomoxetine, the resulting labeledcompound has low affinity for such reuptake sites, and relatively highaffinity for serotonin reuptake sites. In vivo labeling studies haveshown that it is an unacceptably poor probe even for the serotoninreuptake sites because it exhibits low total brain uptake andimmeasurably low specific uptake.

An iodinated compound can be useful as an in vitro probe, but may beuseless as an in vivo probe, because an in vivo probe must meet therequirements associated with intravenous administration of the probe toa living subject. Reasons for the loss of in vivo utility include thefact that the compound may be metabolized too quickly, that it may notcross the blood-brain-barrier, and that it may have high non-specificuptake into the lipid stores of the brain. In vitro homogenate bindingstudies remove these obstacles by isolating the brain tissue fromhepatic metabolic enzymes, by homogenizing the brain tissue so as todestroy the blood-brain-barrier, and by diluting the brain tissue so asto decrease the concentration of lipids in the assay tube. Accordingly,it cannot be assumed that a probe will be useful in both in vivo and invitro modalities.

An in vivo SPECT probe was developed by iodinating cocaine. However,this probe shows a binding affinity and specificity no better thancocaine itself, which is inadequate for purposes of SPECT imaging.

SUMMARY OF THE INVENTION

An iodinated neuroprobe is provided for mapping monoamine reuptakesites. The iodinated neuroprobe is of the formula: ##STR2## wherein Rcan be aryl, substituted aryl, heterocyclic, CO(CF₂)_(n) Y, (CF₂)CHF₂,and (CH₂)_(n) Y. Y can be Cl, Br, I, (CH₂)_(m), aryl, substituted arylheterocyclic, CH₂ H, CO_(R) ³, CO₂ NR³ R⁴, OH, OR³, CH(OR³)₂, CR³(OR⁴)₂, OCOR³, OSO₂ R³, OCONR³ R⁴, OCOOR³, CONR³ R⁴, NR³ R⁴, NR³ COR⁴,NR³ CO₂ R⁴, NR³ CONR⁴ R⁵, NCS, NCO. R³, R⁴ and R⁵ can be alkyl,substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl,or heterocyclic; m=3-8 and n=1-6. R' can be C_(w) H_(2w+1) wherein w=0-6and C includes an isotope of carbon, including at least one radioactiveisotope of carbon. X can be an isotope of Cl, an isotope of Br, anisotope of F, an isotope of I, or Sn(R"₁ R"₂ R"₃) wherein R"₁, R"₂, andR"₃ are C_(p) H_(2p+1) groups where p=1-6, or an aryl group.

For each of the foregoing embodiments there is provided a precursor ofthe radiolabeled neuroprobe that lacks a radiotracer atom, and a kit forpreparing an associated iodinated neuroprobe. Also included arederivatives of the neuroprobes that include ¹⁸ F substituted onto R. Asused herein "β-CIT" and "CIT" refer to N-(N', N'-dimethyl)acetamido-2β-carbomethoxy-3β-(4-iodophenyl) nortropane. As used herein,the substituent (CH₂)_(m) refers to a cycloalkyl group, e.g.,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

Both the radiostable and radioactive variants of the iodinatedneuroprobe of the invention are useful for human and non-human research.For example, in vivo and in vitro experiments can be performed using thecompounds of the invention to study dopamine reuptake sites generally,and cocaine binding sites in particular.

DESCRIPTION OF THE DRAWING

The invention will be more fully understood from the following detaileddescription, in conjunction with the accompanying figures in which:

FIG. 1 shows prior art compounds compared to compounds of the invention;

FIG. 2 shows regional activity in a baboon brain following injection ofa compound of the invention;

FIG. 3 shows a synthesis route for a compound of the invention;

FIG. 4 shows regional areas of brain uptake of a compound of theinvention;

FIG. 5A shows regional activity in a baboon brain following injection ofcompound of the invention; and

FIG. 5B shows regional activity in a baboon brain following injection ofa compound of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Metabolically stable cocaine analogs such as2β-carbomethoxy-3β-(4-iodophenyl)-tropane), an iodine-containing analogof β-CIT (also designated RTI-55), as shown in FIG. 1, compound 3, havehigh affinities for dopamine and serotonin reuptake sites in brain. Aswill be discussed below, ¹²³ I!-β-CIT is shown to be a SPECT (singlephoton emission computed tomography) radiotracer for dopamine andserotonin reuptake sites.

¹²³ I!-β-CIT was prepared by reaction of the corresponding tributyltinprecursor with no-carrier added Na ¹²³ I! in the presence of peraceticacid, followed by preparative HPLC on a C-18 column withmethanol/water/triethylamine (75/25/0.2) at a flow rate of 1.0 ml/min.The final product was formulated in 6 ml sterile saline containing 5-10%ethanol.

Six SPECT experiments were performed in four female baboons (10 kg Papioanubis) under isoflurane anesthesia. The animals were injected with10.6±1.4 mCi ¹²³ I!-β-CIT and scanned for 333±25 min in either the 810XBrain Imager (Strichman Medical Equipment; five experiments) or theASPECT device (Digital Sintigraphics, Cambridge, Mass.; one experiment),with these and subsequent data expressed as means ±S.E.M. Serial 2-6 minimages were reconstructed assuming uniform attenuation equal to that ofwater in an ellipse drawn around the brain. Data were decay-corrected tothe time of injection.

FIG. 2 illustrates regional activity in baboon brain following injectionof 9.6 mCi ¹²³ I!CIT. Activity is expressed in arbitrary units knownfrom phantom studies to be linear with radioactive concentrations. Theactivities in three brain regions are graphed wherein the trace of opencircles is the striatum, the trace of open squares is the midbrain, andthe trace of open diamonds is the cerebellum. The highest activitieswere found in the striatal region and reached peak levels at 179±9 min(n=6) post injection (p.i.)(FIG. 2). Striatal activity was monitored intwo animals for an additional 190 and 260 min post peak values. In oneanimal, striatal activity was virtually unchanged for the remaining 190min of the experiment. With reference to FIG. 2, in the second animal,washout of striatal activity was fit to an exponential function and hadt_(1/2) =27 h (r=0.92).

The brain region which approximately overlay the mesencephalon ormidbrain area had the second highest levels of activity. Midbrain valuespeaked earlier (45±16 min p.i.; n=6) and washed out more rapidly(t_(1/2) =294±59 min; r=0.98±0.01; n=3) than that in the striatum.

At the time of peak striatal uptake, the ratios of regional brainactivities were: striatum (100%); hypothalamus (38.1±5.2%); occipitallobe (13.5±0.8%); temporo-parietal lobes (14.3±2.0%); frontal lobe(10.3±1.0%); and cerebellum (10.0±1.5%), all measured with n=6.

(-)Cocaine (FIG. 1, compound 1) and CFT (FIG. 1, compound 2), bothpotent dopamine and serotonin reuptake inhibitors, induced rapid anddose-dependant displacement of both striatal and midbrain activity.(-)Cocaine (2.9 μmol/kg) administered at 200 min p.i. causeddisplacement of 17% of striatal and 49% of midbrain levels within 30-65min. At 14.7 Fmol/kg administered at 230 min p.i., the correspondingcumulative displacements were 62% and 77%, respectively, within the sameperiod of time.

CFT (0.4 μmol/kg) administered i.v. at 180 min p.i. caused displacementof 57% of striatal and 72% of midbrain levels within 60-120 min. At 2.0μmol/kg administered at 298 min p.i., the corresponding cumulativedisplacements were 83% and 91%, respectively, within the same period oftime.

In contrast, citalopram (a selective serotonin reuptake inhibitor)caused greater displacement of midbrain than striatal activity. At adose of 8.3 μmol/kg i.v. at 190 min p.i., midbrain levels decreased by57% during the following 110 min, compared to only 5% decrease instriatal activity during the same period.

¹²³ I!-β-CIT appears to be a useful SPECT tracer of the dopamine andserotonin reuptake sites. Brain uptake and washout are relatively slowin comparison to cocaine itself and are consistent with themetabolically resistant chemical structure of β-CIT and the location ofthe radioiodine in a chemically stable position. Striatal uptake appearsto largely represent labeling of the dopamine reuptake site, whereasthat in the midbrain is largely associated with the serotonin reuptakesite. The high ratios of striatal to cerebellar activity of ¹²³ I!-β-CITare consistent with low non-specific uptake of the tracer, and suggestthat ¹²³ I!-β-CIT may be a useful clinical marker of dopaminergicdeficiencies in Parkinson's disease.

Referring again to FIG. 1, in a second study (Neumeyer, J. L. et al., J.Med. Chem., 34: 3144-3146 (1991)), the potent cocaine analog2β-carbomethoxy-3β-(4-fluorophenyl)tropane (compound 2) (also referredto as CFT or WIN 35,428 (Clarke, R. L., et al., 1973; Madras, B. K. etal., 1989)) when tritiated or labeled with ¹¹ CH₃ was found to besuperior to ³ H!cocaine or ¹¹ C!cocaine (Fowler, J. S. et al., Synapse4: 371-377 (1989)) as a radioligand probe for cocaine receptors in termsof higher affinity and larger residence time on the dopamine reuptakesite. For further development of analogues suitable for PET and SPECTimaging, 2β-carbomethoxy-3β-(4-iodophenyl) tropane were synthesized andcharacterized (compound 3a; designated as β-CIT in analogy to CFT, itscorresponding, N-demethylated derivative (compound 4; designated asnor-CIT), and the C₂α isomer (compound 3b), as shown in FIG. 1.

Referring to FIG. 3, a synthesis protocol for ¹²³ I!-β-CIT is described.Ecgonidine methyl ester (compound 5) was prepared from cocaine by theprocedure of Clarke et al. (1973). Treatment of compound 5 withphenylmagnesium bromide and subsequent workup with trifluoroacetic acidat low temperature gave a mixture of C₂ epimers (compound 6) (45%) and(compound 7) (31%), which were separated by flash chromatography(silica; CH₂ Cl₂ /CH₃ OH, 25:1). Direct iodination of compound 6 with I₂/HNO₃ /H₂ SO₄ gave the para-substituted compound 3a (β-CIT) as an oil;62%; α!₂₅ D-2.0° (c=0.85, CHCl₃). D-Tartrate salt; mp 72°-74° C.; α!²⁵ D-87.7° (c=1.5, CH₃ OH). Iodination of compound 7 by the same proceduregave compound 3b (α-CIT) as an oil; 39% α!²⁵ D +44° (c=2.5, CHCl₃).1,5-naphthalenedisulfonate salt; mp 139°-140 ° C. N-Demethylation ofcompound 6 was accomplished by conversion to its 2,2,2,-trichloroethylcarbamate followed by reduction (Zn/acetic acid) to yield compound 8 bythe procedure previously described by Milius, R. A., et al., J. Med.Chem. 34 1728-1731 (1991), herein incorporated by reference, followed byiodination to yield nor-CIT (compound 4), which was isolated as a yellowcrystalline solid (free base 48% from compound 6): mp 149°-151° C.; α!²⁵D -67.4° (c=1, CHCl₃).

¹²³ I!-β-CIT (compound ¹²³ I-3a) was synthesized from nonradioactiveβ-CIT (compound 3a) by conversion to the corresponding tributyltinderivative (compound 9). Treatment of compound 3a with bis(tributyltin),tetrakis(triphenylphosphate)palladium(0), and palladium (II) acetate inrefluxing tetrahydrofuran gave compound 9 as a colorless waxy solidafter flash chromatography (silica, stepwise gradient, hexane tohexane/ether, 75:25) in 26% yield from 3a. The 300 MHz NMR (CDCl₃) ofcompound 9 was consistent with the assigned structure. Reaction ofcompound 9 with carrier-free Na¹²³ I in the presence of peracetic acidgave compound ¹²³ I!-3a. The radioiodinated product compound ¹²³ I!-3awas purified by preparative HPLC (Novapak C₁₈, MeOH/H₂ O/Et₃ N,75:25:0.2, 1.0 mL/min; t_(R) 6.7 min) and formulated in normal salinecontaining 5% ethanol an 1% ascorbic acid. Compound ¹²³ I!-3a wasobtained in average overall yield of 60.0±13.4% and with radiochemicalpurity of 97.6±1.6%. The tributyltin precursor used in radiolabelingcontained about 7 mol % CIT carrier, resulting in an ¹²³ I producthaving a specific activity of about 2000 ci/mmol.

The affinities of cocaine (compound 1), α-CIT (compound 3b), β-CIT(compound 3a), and β-CFT (compound 2) for the dopamine and serotoninreuptake sites were determined from radioligand displacement studiesusing tissue homogenates prepared from baboon and rat brain, shown inTable 1 below.

                  TABLE I                                                         ______________________________________                                        In Vitro Radioligand Binding Data                                             for Cocaine and 3-(4-Halophenyl) Analogues.sup.a                                                  displacement                                              displacement of  .sup.3 H!CFT                                                                     of  .sup.3 H!paroxetine                                   analogue                                                                             IC.sub.50 (nM)                                                                          Hill slope (nH)                                                                          IC.sub.50 (nM)                                                                        Hill slope (nH)                           ______________________________________                                        1(cocaine)                                                                           221 ± 14                                                                             0.69 ± 0.06 (3)                                                                       207 ± 66                                                                           0.73 ± 0.12 (5)                        2(β-CFT)                                                                        15.3 ± 1.2                                                                           0.75 ± 0.01 (3)                                                                       479 ± 59                                                                           1.34 ± 0.22 (3)                        3b(α-CIT)                                                                      87.6 ± 2.9                                                                           0.70 ± 0.07 (2)                                                                       210 ± 86                                                                           0.73 ± 0.04 (2)                        3a(β-CIT)                                                                        1.6 ± 0.15                                                                          0.79 ± 0.04 (3)                                                                        3.78 ± 0.53                                                                       0.82 ± 0.08 (6)                        ______________________________________                                    

The data in Table 1 represent radioligand binding of ³ H!CFT (0.5 nM) todopamine reuptake sites in tissue homogenates prepared from primatestriatum and binding of ³ H!paroxetine to serotonin reuptake sites inhomogenates prepared from rat cortical membranes. The IC₅₀ value is theconcentration of displacing analogue required to decrease specificradioligand binding by 50%. Values represent means ±SEM (of nexperiments).

With reference to FIG. 4, five SPECT (single photon emission computertomography) experiments were performed with four female baboons (Papioanubis, 10-12 kg) under isoflurane anesthesia. Animals were injectedi.v. with 8.1±1.4 mCi ¹²³ I!-β-CIT (with these and subsequent dataexpressed as mean ±SEM) and scanned for 300±41 min with the 810X BrainImager (Strichman Medical Equipment, Medfield, Mass.). Serial 1-2 minimages were reconstructed assuming uniform attenuation equal to that ofwater in an ellipse drawn around the brain. Data were decay corrected totime of injection.

FIGS. 5A and 5B illustrate regional activity in baboon brain followingiv injection of 12.1 mCi (FIG. 5A) and 4.2 mCi (FIG. 5B) ¹²³ I!CIT.Activity is expressed in arbitrary units known from phantom studies tobe linear with radioactive concentrations. Displacing agents (FIG. 5A:13 μmol Lu-19-005 per kg; FIG. 5B: 7.4 μmol Citalopram per kg) wereinjected iv at the times marked with arrows. Activities in three brainregions are graphed wherein the trace of filled squares is the striatum,the trace of open circles is the midbrain, and the trace of Xs is thecerebellum. Highest brain uptake overlay the striatal region and peakedat 154±19 min postinjection (pi) of the radioligand and showed striatalto cerebellar ratios at that time of 9.8±1.6. Washout of striatalactivity was followed for an additional 200 and 260 min in two of threecontrol animals and showed 0% and 12% decreases, respectively, from timeof striatal peak to end of the experiment.

With reference to FIGS. 5A and 5B, the brain area with second highestactivities approximately overlays the midbrain and showed peak levels at43±5 min pi (n=5) and had a faster washout than striatal activity.

The pharmacological specificity of the in vivo labeling of ¹²⁵ I!-β-CITwas examined with displacement of brain activity by indatraline (alsodesignated Lu 19-005), a potent agent for the dopamine and serotoninreuptake sites, and citalopram, an agent selective for the serotoninreuptake site. Indatraline (3 μmol/kg iv) injected at 200 min piradioligand caused significant decrease of both striatal and midbrainactivity, as shown in FIG. 5A. During the 100 min period after injectionof Lu 19-005, striatal activity decreased by 65% compared to a meandecrease of 2% during the same period in the two control animalsfollowed for that length of time. In contrast, citalopram (7.4 μmol/kgiv) injected 60 min pi radioligand showed a selective decrease ofmidbrain activity, as shown in FIG. 5B. Citalopram caused a 48% decreaseof midbrain activity during the 60-min period after injection, incomparison to 16±3% decrease (n=3) of midbrain activity in controlanimals followed during this same period.

These results showed that ¹²³ I!-β-CIT was a useful SPECT probe ofmonoamine reuptake sites in primates. The majority of striatal activitywas associated with dopamine reuptake sites, and the majority ofmidbrain activity was associated with serotonin reuptake sites, which isconsistent with the densities of these monoamine transporters measuredin postmortem primate brains. Brain washout of activity was relativelyslow, in part because of the high affinities of β-CIT for the monoaminetransporters. In addition, the iodine atom appears to be in a relativelymetabolically resistant position, since whole body scanning showed lowthyroid uptake, which is indicative of a slow in vivo rate ofdeiodination. ¹²³ I!-β-CIT and ¹¹ C!-β-CIT may be useful clinicalmarkers of dopaminergic and serotonergic innerration in human disorderssuch as Parkinson's disease and depression, which are thought to haveabnormalities in these neuro-transmitter systems.

EXAMPLES OF SYNTHESES Example 1

2-beta-Carbomethoxy-3-beta-(4-iodophenyl)tropane

A mixture of 2-beta-carbomethoxy-3-beta-phenyltropane (See Example 1Abelow and Milius et al. J. Med. Chem., 1991, 34, 1728) (2.9 g, 11.5mmol) and I₂ (3 g. 11.8 mmol) in 25 ml of glacial acetic acid wasstirred and treated dropwise with a mixture of 4.7 mL of concentratednitric acid and 4.7 mL of concentrated sulfuric acid. The reactionmixture was heated to 55° C. and stirred for 2 hours, then cooled toroom temperature and poured onto ice (100 g) and filtered. The pH of thefiltrate was adjusted to 9.5 by the addition of concentrated ammoniumhydroxide at 0°-5° C. The resulting precipitate was removed byfiltration and dissolved in methylene chloride (250 ml). The filtratewas extracted with two 50 mL portions of methylene chloride. Theextracts and solution of precipitate were combined, washed with brine(50 ml) and dried over magnesium sulfate. After the removal of thesolvent, 3.9 g (90.4%) of 2-beta-carbomethoxy-3-beta-4-iodophenyltropanefree base was obtained as an oil.

The free base was dissolved in methanol (20 ml) and combined with 1.5 gof D-(-)tartaric acid in 20 ml of methanol. After the removal ofmethanol under reduced pressure, the residue was recrystallized frommethanol ether (3:1) to give2-beta-carbomethoxy-3-beta-(4-iodophenyl)tropane D-tartrate salt aswhite crystals, m.p. 72°-74° C. C₁₆ H₂₀ NO₂ I.C₄ H₆ O₆. Calculated: C:44.88, H: 4.89, N: 2.62. Found: C: 44.70, H: 4.94, N: 2.57. alpha!_(D)²² =-87.7° (c=0.3, CH₃ OH).

Example 1A

2-beta-Carbomethoxy-3-beta-phenyltropane

A 2M ethereal solution of phenylmagnesium bromide (83 mL, 166 mmol) in a500-mL 3-neck round-bottom flask equipped with mechanical stirrer,addition funnel, and nitrogen inlet tube was diluted with 83 mL ofanhydrous diethyl ether and cooled to -20° C. under an atmosphere of drynitrogen. A solution of anhydroecgonine methyl ester, prepared fromcocaine (1) (15 g, 82.8 mmol) in anhydrous ether (75 mL) was addeddropwise. The heterogeneous mixture was stirred for 1 h at -20° C., thenpoured into an equal volume of ice and water, and acidified by thedropwise addition of 2M HCl. The aqueous layer was made basic by theaddition of concentrated ammonium hydroxide, saturated with NaCl, andextracted with diethyl ether. The combined extracts were dried (Na₂ SO₄)and concentrated in vacuo to give a brown oil. Bulb to bulb distillation(70° C., 0.9 Torr) of the crude product gave a pale yellow oil (16 g,70%). TLC analysis of the oil (silica, pentane/diethylether/2-propylamine, 15:5:0.8) showed it to be a mixture of the C-2alpha and beta epimers. The beta isomer was isolated by silica gelchromatography (pentane: diethyl ether: isopropyl amine, 70:30:3). m.p.63°-66° C. (lit: 62°-64,5° C.: Clarke et al. J. Med. Chem. 16: 1260(1973)).

Example 2

2-alpha-Carbomethoxy-3-beta-iodophenyltropane

The mixture of alpha and beta-2-carbomethoxy-3-beta-iodophenyltropanesprepared as described in Example 1 were separated by silica gelchromatography as described in Example 1. Fractions containing thealpha-2-carbomethoxy-3-beta-iodophenyltropane were pooled andconcentrated in vacuo. The free base thus obtained was treated withnaphthalene-1,5-disulfonic acid. The crude salt was recrystallized fromacetonitrile to give the 2-alpha-carbomethoxy-3-beta-iodophenyltropanenaphthalene-1,5-disulfonate salt, m.p. 166°-168° C. C₁₆ H₂₀ NO₂ I•C₁₀ H₆(SO₃ H)₂ •2H₂ O. Calculated: C: 40.01, H: 4:55, N: 1.97, I: 17.90;Found: C: 43.94, H: 4.55, N: 1.91, I: 17.99.

Example 3

2-beta-Carbomethoxy-3-beta-(4-iodophenyl)nortropane

A solution of2-beta-carbomethoxy-3-beta-(4-iodophenyl)tropane (410 mg,1.5 mmol) in toluene (20 mL) was treated with of 2,2,2-trichloroethylchloroformate (1 mL, 7.3 mmol). The mixture was heated at 120° C. for 1hour, cooled to room temperature, and evaporated to dryness in vacuo.The residue was partitioned between methylene chloride and water. Theorganic layer was separated, dried (N₂ SO₄), and concentrated in vacuoto give the trichloroethyl chloroformate as a dry foam. The crudecarbamate was dissolved in 50% aqueous acetic acid, treated with 200 mg(0.0067 g-atom) of zinc dust, and stirred at room temperature for 16hours. The reaction mixture was filtered adjusted to pH 7 withconcentrated ammonium hydroxide, saturated with NaCl, and extracted withdiethyl ether. The extracts were combined, dried (Na₂ SO₄), andconcentrated in vacuo. The residue was purified by flash chromatography(silica, pentane/diethyl ether/isopropylamine, 3:7:0.7) to afford2-beta-carbomethoxy-3-beta-(4-iodophenyl)nortropane, which was isolatedas a yellow crystalline solid, m.p 149°-151° C.; alpha!²⁵ _(D) -67 4°(c=1, CHCl₃).

Example 4

2-beta-Carbomethoxy-3-beta-(4-iodophenyl)-8-(3-fluoropropyl)-nortropane

A solution of 2-beta-carbomethoxy-3-beta-(4-iodophenyl)-nortropane (371mg, 1.0 mmol), 1-bromo-3-fluoropropane (155 mg, 1.1 mmol), andtriethylamine (0.5 mL) in dry toluene (20 mL) was stirred under anatmosphere of dry nitrogen and heated to reflux. After four hours, thereaction mixture was cooled to room temperature and filtered. Thefiltrate was concentrated under reduced pressure, and the residuechromatographed on a silica column (eluant: diethyl ether).Concentration of product-containing fractions gave2-beta-carbomethoxy-3-beta-(4-iodophenyl)-8-(3-fluoropropyl)nortropaneas a white solid, m.p. 78.5°-79.5° C. C₁₈ H₂₃ NO₂ FI, Calculated: C:50.13, H: 5.34, N: 3.25; Found: C: 50.27, H: 5.26, N: 3.15.

Example 5

2-beta-Carbomethoxy-3-beta-(3-fluoro-4-iodophenyl)tropane

A mixture of 2-beta-carbomethoxy-3-beta-(3-fluorophenyl) tropane (400mg, 1.44 mmol), silver sulfate (400 mg, 1.3 mmol), iodine (600 mg, 2.36mmol) and 80% sulfuric acid (9 Ml) was stirred for five days at roomtemperature. The reaction mixture was poured into 150 mL of ice andwater, made basic by the addition of concentrated ammonium hydroxide,and extracted with three 60 mL portions of chloroform. The combinedextracts were washed sequentially with solutions of 10% sodiumbisulfite, 5% sodium carbonate and water, then dried over sodiumsulfate, and filtered. The filtrate was concentrated in vacuo and theoily residue was redissolved in chloroform and treated with a solutionof p-toluene sulfonyl chloride in chloroform. The resulting solid wasrepeatedly recrystallized from water and ethanol to give2-beta-carbomethoxy-3-beta-(3-fluoro-4-iodophenyl)tropanetosylate saltas a white crystalline solid, m.p. 68°-70° C. (soften, 45° C.), C₁₆ H₁₉FINO₂ •C₇ H₈ SO₃ •H₂ O: Calculated: C: 46.55,H: 4.93, N: 2.36; Found: C:46.34, H: 4.86, N:1.99.

Example 6

2 -beta-Carboxy-3-beta-(4-iodophenyl)tropane

A suspension of 2-beta-carbomethoxy-3-beta- (4-iodophenyl)tropane (100mg, 0.26 mmol) in 2 mL of H₂ O was heated at reflux for 10 hours. Theresulting solution was cooled to room temperature, and the resultingprecipitate was collected by filtration and dried under vacuum overnightto give 70 mg (70%) of 2-beta-carboxy-3-beta-(4-iodophenyl)tropane m.p.299°-300° C. C₁₅ H₁₈ NO₂ I.0.5 H₂ O: Calculated C: 47.51, H:5.05, N:3.69: Found: C: 47.28, H: 4.84, N: 3.69.

Example 7

2-beta-Carbomethoxy-3-beta-benzyloxytropane

A stirred suspension of benzyl bromide (3.0 g, 0.015 mol) and potassiumiodide (3.0 g, 0.021 mol) in acetone (20mL) was treated dropwise with asolution of ecgonine methyl ester (2.6 g, 0.014 mol) in acetone (10 mL)at room temperature. The mixture was stirred at room temperature for 70hours, then heated to reflux and stirred for an additional 8 hours. Thereaction mixture was cooled to room temperature and filtered. Thefiltrate was concentrated in vacuo, the residue dissolved in chloroform(200 mL) and extracted with four 50 mL portions of 2N hydrochloric acid.The combined extracts were made basic by the addition of concentratedammonium hydroxide. The resulting mixture was extracted with four 20 mLportions of chloroform. The extracts were dried over sodium sulfate andconcentrated in vacuo to give 1.7 g of2-beta-carbomethoxy-3-beta-benzyloxytropane as an oil.

The product was dissolved in acetonitrile (20 mL) and treated with asolution of naphthalene-1,5-disulfonic acid (2.2 g) in acetonitrile (20mL). The solution was concentrated in vacuo to a syrup, which wasdiluted with diethyl ether. The resulting precipitate was collected byfiltration and dried to give 1.6 g of2-beta-carbomethoxy-3-beta-benzyloxytropane naphthalene-1,5-disulfonatesalt, m.p. 126°-130° C., C₁₇ H₂₃ NO₃.C₁₀ H₆ (SO₃ H)₂.2.5 H₂ O. Elementalanalysis: Calculated, C: 52.08, H: 5.83, N: 2.25. Found, C: 52.02, H:5.69, N: 2.72. alpha!_(D) ²⁴ =-25.4° (c=1, CH₃ OH).

Example 8

2-beta-Carbomethoxy-3-beta-(4-tributylstannylphenyl)tropane

A mixture of 2-beta-carbomethoxy-3-beta-(4-iodophenyl)tropane (250 mg,0.65 mmol), bis (tributyl) distannane (522 mg, 0.9 mmol), tetrakis(triphenylphosphine)palladium(0) (3 mg) and anhydrous toluene (10 mL)was heated to reflux under an atmosphere of dry nitrogen and stirred for28 hours. The mixture was filtered, and the filtrate concentrated invacuo. The residue was applied to a silica gel column and eluted with amixture of hexane:diethyl ether:isopropyl amine (70:30:3). The fractionscontaining product were pooled, concentrated in vacuo and treated withpentane to precipitate2-beta-carbomethoxy-3-beta-(4-tributylstannylphenyl)tropane as a solid.The 300 MHz NMR spectrum was consistent with the assigned structure.alpha!_(D) ²² =-8.9° (c=0.4, CHCl₃).

Example 9

¹²³ I!-2-beta-Carbomethoxy-3-beta-(4-iodophenyl)tropane

To a vial containing 50 μg (0.094 μmol) of2-beta-carbomethoxy-3-beta-(4-tributylstannylphenyl)tropane was added 50μL ethanol, 150 μL 0.5M H₃ PO₄, 125-500 μL (20-30 mCi) ¹²³ I!NaIsolution, and 100 μL (4.2 μmol) 0.042M peracetic acid. After 20-30minutes, 50 μL of 100mg/mL aqueous NaHSO₃ solution was added. SaturatedNaHCO₃ solution was added, and the mixture extracted with ethyl acetate.The combined extracts were dried (Na₂ SO₄) and concentrated to dryness.The residue was redissolved in methanol and purified by HPLC (C-18column, eluant:CH₃ OH:H₂ O:triethylamine; 75:25:0.2). The fractioneluting at the retention time of2-beta-carbomethoxy-3-beta-(4-iodophenyl)tropane was collectedevaporated to dryness and reconstituted in 5% ethanol and 0.1 nMascorbic acid.

In SPECT applications, the radiostable iodinated neuroprobe of theinvention is useful as a reference standard, and can also be used as adilutant for the radioactive form of the neuroprobe. The radioiodinatedcompound is generally identified by its chromatographic mobility ascompared with a fully characterized reference standard. Thus,preparation of the radioiodinated compound requires the non-radioactiveiodinated compound.

To avoid the necessity of storing a radioactive neuroprobe, it is usefulto provide a kit containing the non-radioactive iodinated compound andan appropriate oxidizing agent, such as perchloric acid, performic acid,peracetic acid, hydrogen peroxide, hydrogen peroxide withlactoperoxidase, 1,3,4,6-tetrachloro-3α,6α-diphenylglycouril, or aN-chloro-4-methylbenzenesulfonamide sodium salt. Then, thenon-radioactive precursor compound can be oxidized in the presence of asuitable radioactive compound, such as the carrier free Na ¹²³ I! shownin the synthesis route described herein, any other radioisotope source,such as any solution of a salt of a radioactive isotope of iodine, areagent containing ^(m) C_(n) H_(2n+1) X, where n=0-6 and X is a leavinggroup, or a reagent containing ¹⁸ F of the formula FC_(n) H_(2n) X,where n=0-6 and X is a leaving group, to prepare the iodinatedneuroprobe at its time and place of use.

Radiolabeled neuroprobes of the invention are also useful in otherimaging procedures. For example, an ¹²⁵ I-labeled neuroprobe can be usedin autoradiography or therapy, and an ¹³¹ I-labeled neuroprobe is usefulas a multiple photon emitter for use in animal studies. Also, ¹¹ C-, ¹⁴C-, and ¹⁸ F-labeled neuroprobes can be used in PET imaging.

Both the radiostable and radioactive variants of the iodinatedneuroprobe of the invention are useful for human and non-human research.For example, in vivo and in vitro experiments can be performed using thecompounds of the invention to study the dopamine transporter generally,and cocaine binding sites in particular.

Additionally, the radiostable version of the neuroprobe of the inventioncan be used as a drug for influencing dopamine reuptake.

In an alternative embodiment, an intermediate is provided that includesfunctional moieties attached to N-8. Such moieties include aryl,substituted aryl, heterocyclic, CO(CH₂)_(n) Y, (CF₂)_(n) CHF₂, and(CF₂)_(n) Y, wherein Y=Cl, Br, I, (CH₂)_(m), aryl, substituted aryl,heterocyclic, CO₂ H, CO₂ R³, CO₂ NR₃ R⁴, OH, CH(OR³)₂, CR³ (OR⁴)₂,OCOR³, OSO₂ R³, OCONR³ R⁴, OCOOR³, CONR³ R⁴, NR³ R⁴, NR³ COR⁴, NR³ COR⁴,NR³ CONR⁴ R⁵, NCS, NCO; R³, R⁴ and R⁵ =alkyl, substituted alkyl,alkenyl, substituted alkenyl, aryl, substituted aryl, or heterocyclic;m=3-8; and n=1-6.

In one embodiment, the functional substituents of N-8 include leavinggroups, such as halogens, carboxylate esters or sulfonate esters.Sulfonate esters, such as mesylates, tosylates and triflates(trifluoromethanesulfonates) are particularly useful leaving groups.Other groups may be substituted at this position for the purpose ofenhancing or reducing lipophilicity, to permit further chemicalmodification, or to provide a site for biological transformations, suchas alkylation, reduction or oxidation. These groups include esters,amides, ethers, acetals, ketals, carbamates, carbonates, amines, ureas,isothiocyanates, phthalamidoalkyl, (N', N'-dimethyl)acetamido,2,2-di,ethoxyethyl, carbomethoxymethyl, aryl, substituted aryl,heterocyclic, tetrahydropyran, cycloalkymethyl, and the like.

General Procedure for N-Alkylation of Nor-β-CIT

N-Alkylation reactions are typically carried out with 0.27 mmol ofnor-β-CIT (compound 4). To a solution of nor-β-CIT and triethylamine (46mmol) in absolute EtOH or anhydrous toluene is added the appropriatealkyl bromide (0.4 mmol) and KI (10 mg). The mixture is refluxed undernitrogen from 1 to 24 hours depending on alkyl bromide monitoring bythin layer chromatography (TLC) to the completion of reaction. Thesolvent is then removed under reduced pressure and the residue is passedthrough a silica gel column (eluted with hexane/ether/TEA) to yield thepure compounds. Examples 10-14 describe alkylation reactions of the N-8group.

Example 10

Synthesis of N-phthalamidopropyl-β-CIT

Nor-β-CIT in triethylamine is combined with phthalamidopropyl bromideaccording to the protocol set forth above. The product,N-phthalamidopropyl-β-CIT, has the following physical characteristics:mp 136°-138° C. (HCl salt); α!_(D) ²⁰ -119.8° C. (C, 0.31, MeOH) (freebase).

¹ H NMR (250 MHz, CDCl₃) δ 7.83 (m, 2H); 7.70 (m, 2H); 7.55 (d, J=8.4Hz, 2H); 7.00 (d, J=8.4 Hz, 2H); 3.79 (m, 1H); 3.68 (m, 1H); 3.52 (s,3H); 3.41 (m, 1H); 2.89 (m, 2H); 2.51 (m, 3H); 2.32 (m, 3H); 2.03 (m,2H); 1.67 (m, 5H).

MS (FAB, NBA): 559 (27%); 445 (22%); 444 (100%); 417 (27%); Anal. (C₂₆H₂₆ N₂ O₄ I•HCl•H₂ O; Calcd. C, 51.04; H, 4.78; N, 4.58; Found C, 50.99;H, 4.92; N, 4.54.

Example 11

Synthesis of N-((N', N'-dimethyl)acetamido)-β-CIT

Nor-β-CIT in triethylamine is combined with N',N'-dimethylbromoacetamideaccording to the protocol set forth above. The product,N-((N',N'-dimethyl)acetamido)-β-CIT, has the following physicalcharacteristics: mp 194°-196° C.; α!_(D) ²⁰ -45.3° (c, 0.3, MeOH).

¹ H NMR (250 MHz, CDCl₃): δ 7.58 (d, J=8.3Hz, 2H); 7.00 (d, J=8.3 Hz,2H); 3.70 (m, 1H); 3.45 (s, 3H); 3.12 (m, 1H); 3.11 (m, 2H); 2.90 (s,3H); 2.55 (m, 1H); 2.18 (m, 2H); 1.65 (m, 4H); Anal. (C₁₉ H₂₄ N₂ O₃ I);Calcd. C, 50.12; H, 5.31; N, 6.15; Found C, 49.88; H, 5.60; N, 6.04.

Example 12

Synthesis of N-(2,2-dimethoxyethyl-β-CIT

Nor-β-CIT in triethylamine is combined with 2,2,-dimethoxyethyl bromideaccording to the protocol set forth above. The product,N-(2,2-dimethoxyethyl)-β-CIT, has the following physicalcharacteristics: mp 126°-128 ° C.; α!_(D) ²⁰ -36.6° (c, 0.3, MeOH).

¹ H NMR (250 MHz, CDCl₃) δ 7.66 (d, J=8.3 Hz, 2H); 7.02 (d, J=8.3 Hz,2H); 4.32 (t, J=5.2 Hz, 1H); 4.48 (m, 1H); 3.78 (m, 1H); 3.51 (s, 3H);3.42 (m, 1H); 3.37 (s, 3H); 3.35 (s, 3H); 2.88 (m, 2H); 2.57 (td, J=2.7Hz, J=12.1 Hz; 1H); 2.41 (m, 2H); 2.03 (m, 2H); 1.66 (m, 4H).

MS (FAB,NBA): 461 (21%); 460 (100%, M+H⁺); 459 (2%); 428 (12%); 245(23%); Anal. (C₁₉ H₂₆ NO₄ I); Calcd. C, 49.68; H, 5.71; N, 3.05; FoundC, 49.71; H, 5.71; N, 2.99.

Example 13

Synthesis of N-(carbomethoxymethyl)-β-CIT

Nor-β-CIT in triethylamine is combined with carbomethoxymethyl bromideaccording to the protocol set forth above. The product,N-(carbomethoxymethyl-β-CIT, has the following physical characteristics:mp 120°-122° C.; α!_(D) ²⁰ -58.7° (C, 0.3, MeOH).

¹ H NMR (250 MHz, CDCl₃) δ: 7.58 (d, J=8.4 Hz, 2H); 7.02 (d, J=8.4 Hz,2H); 3.74 (m, 1H); 3.68 (s, 3H); 3.51 (s, 3H); 3.58 (s, 3H); 3.45 (m,1H); 3.14 (dd, J=16.5 Hz, J=13.3 Hz, 2H); 2.90 (m, 2H); 2.75 (t, J=9.8Hz, 1H); 2.12 (m, 1H); 2.01 (m, 1H); 1.68 (m, 3H).

MS (FAB, NBA): 445 (20%); 444 (100%, M+H⁺); 443 (16%); 412 (5%); 385(9%); 384 (45%); Anal. (C₁₈ H₂₂ NO₄ I); Calcd. C, 48.77; H, 5.00; N,3.18; Found C, 48.63; H, 5.05; N, 3.12.

Example 14

Synthesis of N-(cyclopropylmethyl)-β-CIT

Nor-β-CIT in triethylamine is combined with cyclopropylmethyl bromideaccording to the protocol set forth above. The product,N-(cyclopropylmethyl)-β-CIT, has the following physical characteristics:mp 75°-77° C.; α!_(D) ²⁰ -27 6° (c 0 3, MeOH).

¹ H NMR (250 MHz, CDCl₃): δ 7.57 (d, J=8.4 Hz, 2H); 7.02 (d, J=8.4 Hz,2H); 3.95 (m, 1H); 3.59 (s, 3H); 3.43 (m, 1H); 3.58 (s, 3H); 2.90 (m,2H); 2.55 (dd, J=12.1 Hz, J=2.8 Hz, 1H); 2.39 (dd, J=12.3 Hz, J=5.3 Hz,1H); 1.96 (m, 3H); 1.64 (m, 4H); 0.78 (m, 1H); 0.43 (m, 2H); 0.06 (m,2H).

MS (FAB, NBA): 427 (25%); 426 (100%, M+H⁺); 425 (8%); 424 (11%); 300(8%); Anal. (C₁₉ H₂₃ NO₂ I); Calcd. C, 53.78; H, 5.46; N, 3.30; Found C,53.58; H, 5.67; N, 3.26.

Example 15

Synthesis of8-(3-chloropropyl)-2β-carbomethoxy-3β-(4'-iodophenyl)-8-azabicyclo-3.2.1!octane

N-(3-Hydroxypropyl)nor-β-CIT (1.8 g, 4.2 mmol) is dissolved in methylenechloride (150 ml) and cooled to 0° C. in an ice-bath under nitrogen.Methanesulfonylchloride (580 mg, 4.4 mmol) is added, followed byaddition of 2,6 lutidine (1 mL). The reaction mixture is stirred 2 h andthen a second portion of methanesulfonylchloride (580 mg) is added. Themixture is allowed to come to room temperature and stirred for anadditional 48 h. The solvent is removed and the residue ischromatographed on silica gel column eluting with ether/hexane/TEA(50/50/5) to give 1.4 g of white solid (top 96°-98° C.).

¹ H NMR (300 MHz, CDCl₃): δ 7.58 (d, J=8.4 Hz, 2H); 7.00 (d, 8.4 Hz,2H); 3.75 (m, 7H); 2.95 (m, 2H); 2.57 (dd, 1H); 2.38 (t, 2H); 1.85 (m,7H).

MS (FAB, NBA): 495 (19%); 494 (94%); 493 (33%).; 492 (100%); 491 (14%);490 (7%); 412 (21%); 394 (9%); Anal. C₁₈ H₂₃ C1NO₂ I); Calcd. C, 43.99;H, 4.72; N, 2.85; Found C, 44.10; H, 4.80; N, 2.81.

Example 16

Synthesis of8-(3-bromopropyl)-2β-carbomethoxy-3β-(4'-iodophenyl)-8-azabicyclo-3.2.1!octane

At 0° C., triphenylphosphine (148 mg, 0.55 mmol) is dissolved inmethylene chloride, and bromine (88 mg, 0.55 mmol) is added dropwise.After 10 min, N-(3-Hydroxypropyl)nor-β-CIT (215 mg, 0.5 mmol) is slowlyadded; 30 min later, the solvent is removed at reduced pressure andresidue is passed through a silica gel column eluting with ether to give42 mg of white solid.

¹ H NMR (300 MHz, CDCl₃): δ 7.58 (d, J=8.4 Hz, 2H); 7.00 (d, J=8.4 Hz,2H); 3.75 (m, 7H); 2.95 (m, 2H); 2.57 (dd, 1H); 2.38 (t, 2H); 1.85 (m,7H).

¹³ C NMR (CDCl₃): 171.57; 136.73; 129.33; 90.95; 63.19; 61.16; 52.28;50.95; 50.17; 45.86; 42.81; 39.26; 33.70; 31.70; 25.79; 8.49.

MS (GC/MS): 447; 384; 346; 257; 217; Anal. (C₁₈ H₂₃ BrNO₂ I); Calcd. C,48.29; H, 5.18; N, 3.13; Found C, 48.39; H, 5.19; N, 3.14.

Example 17

Synthesis of8-(2-hydroxyethyl)-2β-carbomethoxy-3β-(4'-iodophenyl)-8-azabicyclo-3.2.1!octane

Nor-β-CIT (5 mmol) is dissolved in ethanol (30 mL), together with2-bromoethyltetrahydropyran (7.5 mmol), triethylamine (0.76 g) andpotassium iodide (250 mg). The mixture is heated at reflux undernitrogen for 16 h. When the reaction is completed, the solvent isremoved at reduced pressure and the residue is passed through a silicagel column eluting with hexane/ether/triethylamine (15/80/5). Thefractions containing product are collected and concentrated to give thepure protected compound. This compound is stirred with H₂ O (10 mL), THF(10 ml) and acetic acid (30 mL) during 20 h at 60° C. The solvent isremoved, and the residue is basified with NH₄ OH and extracted withdichoromethane. The organic layer is dried over MgSO₄ and concentrated.The residue is passed through a silica gel column eluting withhexane/ether/triethylamine (10/80/10). The fractions containing productare collected and concentrated to give 1.3 g of product as a colorlessoil.

¹ H NMR (300 MHz, CDCl₃): δ 7.58 (d, J=8.4 Hz, 2H); 6.99 (d, J=8.4 Hz,2H); 3.50 (m, 4H); 3.49 (s, 3H); 2.94 (m, 1H); 2.88 (m, 1H); 2.63 (m,2H); 2.42 (m, 2H); 2.05 (m, 2H); α!_(D) ²⁰ -34.06° (c, 0.3, MeOH).

Example 18

Synthesis of N-3-(p-Tolylsulfonyloxyropyl)!-2β-carbomethoxy-3β-(4'-iodopheny)nortropane

A solution of N-(3-hydroxypropyl)nor-β-CIT (150 mg, 0.35 mmol), pyridine(100 mg), and p-toluenesulfonyl chloride (100 mg) in chloroform (15 ml)is stirred at room temperature for 4 hr, diluted with water (50 ml), andextracted with chloroform (100 ml). The organic layer is concentratedunder reduced pressure. The residue is purified by flash chromatographyon silica gel, eluting with hexane/ether/TEA (10/70/0.1) to give 51 mgof product as an oil. The yield is approximately 25%.

¹ H NMR (CDCl₃): δ 1.62 -1.80 (m, 3H), 2.01-2.18 (m, 3H) , 2.45 (s, 3H),2.62 (m, 1H), 2.91 (m, 1H), 3.43 (m, 1H), 3.51 (s, 3H), 3.80 (m, 1H),4.36-4.52 (m, 2H), 6.99-7.58 (ABq, 4H), and 7.55-7.80 (ABq, 4H).Elemental analysis calculated for C₂₅ H₃₀ NO₅ IS•1/2H₂ O: C, 50.68; H,5.27; N, 2.36; Found: C, 50.64; H, 5.45; N, 2.10.

Example 19

Synthesis ofN-(2,2-difluoroethyl)-2β-carbomethoxy-3β-(4'-iodopheny)nortropan

A solution of nor-β-CIT (300 mg, 0.8 mmol),1,1-difluoro-2-trifluoromethanesulfonyloxyethane (300 mg, 1.4 mmol), andtriethylamine (1 ml) in acetone (15 ml) is stirred at room temperatureovernight. The reaction mixture is filtered and the separated residuewashed with toluene (2×2 ml). The combined filtrate and washings areconcentrated under reduced pressure. The residue is purified by flashchromatography on silica gel, eluting with hexane/ether/TEA (10/7/0.1)to give 160 mg of product as a white solid. mp. 113°-114° C.; yield isapproximately 46%.

¹ H NMR (CDCl₃): δ 1.62-1.80 (m, 3H), 2.01-2.18 (m, 3H), 2.53-2.55 (m,2H), 2.62 (m, 1H), 2.91 (m, 1H), 3.43 (m, 1H), 3.51 (s, 3H), 3.80 (m,1H), 4.36-4.52 (m, 1H), 6.99-7.02 and 7.55-7.58 (ABq, 4H). Elementalanalysis calculated for C₁₇ H₂₀ NO₂ IF₂ •1/2H₂ O: C, 45.96; H, 4.77; N,3.22; Found: C, 46.05; H, 4.72; N, 3.16.

Example 20

Synthesis ofN-(3-hydroxypropyl)-2β-carbomethoxy-3β-(4'-iodopheny)tropane

A solution of nor-β-CIT (250 mg, 0.67 mmol), 3-bromopropanol (300 mg,2.13 mmol) and triethylamine (0.5 ml) in toluene (20 ml) is refluxedunder a dry nitrogen atmosphere for 4 hr, cooled and filtered. Theseparated residue is washed with toluene (2×2 ml). The combined filtrateand washings are concentrated under reduced pressure. The residue waspurified by flash chromatography on silica gel, eluting withhexane/ether/TEA (10/7/0.1) to give 168 mg of product as a liquid. Yieldis approximately 58%.

¹ H NMR (CDCl₃): δ 1.62-1.80 (m, 5H), 1.98-2.18 (m, 2H), 2.36-2.42 (m,2H), 2.51-2.63 (m), 2.90-3.02 (m, 2H), 3.40 s(br), m, 1H!, 3.70 s(br),1H!, 4.44-4.59 (m, 2H), 7.00-7.03 and 7.57-7.60 (ABq, 4H). Elementalanalysis calculated for C₁₈ H₂₄ NO₃ F: C, 50.36; H, 5.64; N, 3.26;Found: C, 50.35; H, 5.57; N, 3.19.

Example 21

Synthesis of N-3-(methanesulfonyloxy)propyl!-2β-carbomethoxy-3β-(4'-iodopheny)nortropane

To a solution of N-(3-hydroxypropyl)nor-β-CIT (380 mg, 0.88 mmol) and2,6-lutidine (150 μl) in chloroform (25 ml) is added methanesulfonylchloride (152 mg, 1.33 mmol) at 0° C. The solution is stirred at 0° C.for 2 hr and then a second portion of methanesulfonyl chloride is addedand stirring is continued at room temperature for an additional 4 hr.After removal of the solvent, the residue is purified by flashchromatography on silica gel, eluting with hexane/ether/TEA (10/7/0.1)to give 190 mg of product as an oil. Yield is approximately 40%.

¹ H NMR (CDCl₃): δ 1.62-180 (m, 3H), 2.01-2.18 (m, 3H), 2.45 (s, 3H),2.62 (m, 1H), 2.91 (m, 1H), 3.04 (s, 3H), 3.43 (m, 1H), 3.51 (s, 3H),3.80 (m, 1H), 4.36-4.31 (m, 2H) and 6.99-7.58 (ABq, 4H).

Elemental analysis calculated for C₁₉ H₂₆ NO₅ IS 1/2H₂ O: C, 43.43; H,5.37; N, 2.67; Found: C, 43.12; H, 5.15; N, 2.58.

Preparation of Radiolabeled Compounds from the NonradioactiveIntermediates

In general, the leaving group attached to the moiety at N-8 is capableof being displaced by a radionuclide such as ¹⁸ F. The chemistry of thereaction is based on nucleophilic substitution of ¹⁸ F!fluoride into anactivated precursor dissolved in a solvent.

In general, the precursor is an N-substituted β-CIT derivative thatincludes a leaving group on the moiety attached to N-8. The leavinggroup is preferably a mesylate, or another sulfonate ester, such astosylate, triflate, or a halogen (iodide, bromide, chloride), howeverother leaving groups may also be used. Solvents used in the reaction arepreferably anhydrous, polar, aprotic solvents, such as acetonitrile,dimethylsulfoxide, dimethylformamide, N-methylpyrrolidone,hexamethylphosphoric triamide, and the like.

The radioisotope is generated in minute quantities and generallyrequires an auxilliary reagent to dissolve in the solvent andparticipate in the chemical reaction. The solubilizing agent can be anyagent capable of solubilizing radionuclides that takes the form M^(+X)⁻. M⁺ is preferably the complex of potassium and4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo 8.8.8!hexacosane or an alkalimetal ions, such as Na⁺, Ce⁺, Ru⁺, or a tetraalkylammonium, such astetramethylammonium, or an ion exchange resin functionalized withquaternary amine groups. X⁻ is preferably carbonate, bicarbonate,hydroxide, or formate. However other counterions may also be used.

Example 22

Preparation of ¹⁸ F!-8-(3-fluoropropyl)-2β-CIT fromN-(3-mesyloxypropyl)-N-nor-β-CIT

An aqueous solution of ¹⁸ F!fluoride ions (0.5 mL) is mixed with4,7,13,16,21,24-hexaoxa-l,10-diazabicyclo 8.8.8!hexacosane (10 mg) andpotassium carbonate (1 mg) in a borosilicate glass vessel of 5 mLcapacity. The vessel is partially immersed in an oil bath thermostatedto 100° C., and the solution evaporated to dryness using a stream ofnitrogen. An aliquot of anhydrous acetonitrile (1 mL) is added to thereaction vessel and allowed to evaporate under the nitrogen flow. Thisaddition/evaporation step is performed a second time. Heating of thevessel is continued for approximately one minute after evaporation ofthe second aliquot.

The vessel is next raised above the oil bath. To the residue is added asolution of N-(3-mesyloxypropyl)-N-nor-β-CIT (2 mg; see Example 21) inanhydrous acetontrile (1 mL). The vessel is re-immersed in the oil bathso that the solvent achieves gentle reflux. Heating is continued forapproximately five minutes, and then the vessel is cooled to roomtemperature.

The reaction mixture is concentrated to near dryness under a stream ofnitrogen. The residue is dissolved in 3:1 methanol-water (0.5 mL) andinjected into a high pressure liquid chromatograph fitted with a 10×250mm column packed with octadecyl-functionalized silica and eluted with3:1 methanol-water at 4 mL/min. The effluent is collected in test tubesat 0.5 minute intervals. The fractions containing N-(3- ¹⁸F!fluoropropyl)-N-nor-β-CIT are combined, evaporated to dryness, andredissolved in USP sodium chloride injection containing 5% by volume USPethanol and 0.1 mM L-ascorbic acid.

Other modifications and implementations will occur to those skilled inthe art without departing from the spirit and the scope of the inventionas claimed. Accordingly, the above-description is not intended to limitthe invention except as indicated in the following claims.

What is claimed is:
 1. A neuroprobe for mapping monoamine reuptakesites, the neuroprobe having the formula: ##STR3## wherein R=(CH₂)_(n)OSO₂ R³ ;wherein R³ =alkyl, substituted alkyl, alkenyl, substitutedalkenyl, aryl, substituted aryl, and heterocyclic; and n=1-6; R'=C_(w)H_(2w+1) wherein w=0-6 and C includes an isotope of carbon; and X=anisotope of Cl, an isotope of Br, an isotope of F, an isotope of I, orSn(R"₁ R"₂ R"₃), whereinR"₁ =a C_(p) H_(2p+1) group where p=1-6, or anaryl group; R"₂ =a C_(p) H_(2p+1) group where p=1-6, or an aryl group;and R"₃ =a C_(p) H_(2p+1) group where p=1-6, or an aryl group.
 2. Theneuroprobe of claim 1, wherein the iodine atom is a radioactive isotopeof iodine.
 3. The neuroprobe of claim 2, wherein the radioactive isotopeof iodine is selected from the group consisting of ¹²³ I, ¹²⁵ I, and ¹³¹I.
 4. The neuroprobe of claim 1, wherein in R', C includes a radioactiveisotope of carbon.
 5. A kit for preparing a radiolabeled neuroprobe formapping monoamine reuptake sites, the kit comprising:a precursor of theformula: ##STR4## wherein R=(CH₂)_(n) OSO₂ R³ ;wherein R³ =alkyl,substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl,and heterocyclic; and n=1-6; R'=C_(w) H_(2w+1) wherein w=0-6 and Cincludes an isotope of carbon; and X=an isotope of Cl, an isotope of Br,an isotope of F, an isotope of I, or Sn (R"₁ R"₂ R"₃) whereinR"₁ =aC_(p) H_(2p+1) group where p=1-6 or an aryl group; R"₂ =a C_(p) H_(2p+1)group where p=1-6 or an aryl group; and R"₃ =a C_(p) H_(2p+1) groupwhere p=1-6, or an aryl group.
 6. The kit of claim 5, wherein saidisotope of I is a radioactive isotope of iodine.
 7. The kit of claim 6,wherein said isotope of I is selected from the group consisting of ¹²³I, ¹²⁵ I, and ¹³¹ I.
 8. The kit of claim 6, wherein the precursor isreacted in the presence ¹⁸ F.
 9. The kit of claim 8, wherein the ¹⁸ F isselected from the group consisting of Na¹⁸ F, K¹⁸ F, and Cs¹⁸ F.
 10. Thekit of claim 8, wherein said ¹⁸ F is complexed with a reagent of theformula M⁺ X⁻, wherein:M⁺ =a complex of4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo 8.8.8!hexacosane and one of K,Na, Ce, Ru, a tetraalkylammonium ion, and an ion exchange resinfunctionalized with quaternary amine groups; and X⁻ =carbonate,bicarbonate, hydroxide, and formate.
 11. The kit of claim 5, wherein inR', C includes a radioactive isotope of carbon.